In scanning microscopy, at least one illumination beam is focused by an objective onto a sample. To guide the illumination beam in a scanning or sampling movement across the sample, a scanning element (for example one or more movable mirrors, an AOD, i.e. an acousto-optical deflector, or the like) is arranged upstream of the objective, which scanning element deflects the illumination beam such that this beam performs the required scanning movement on the sample. The scanning element usually comprises one or more mirrors, the tilting movement of which is converted by the optical imaging into a lateral movement of the light spot generated by the illumination beam on the sample. The focused illumination beam thus scans the sample point by point. The detection light emanating from the sample is then detected for each scanned point. Finally, the detection signal thus acquired is composed into an image in a computing unit.
In the field of scanning microscopy, confocal microscopy is a particularly preferred microscopy method. The basic mode of operation of this microscopy method will be explained in the following with reference to FIG. 1 which shows, purely schematically, a confocal microscope which is generally denoted by reference numeral 10.
The confocal microscope 10 has a light source which emits an illumination beam 12 onto a dichroitic beam splitter mirror 14. The beam splitter mirror 14 is configured so that it lets through light of the wavelength of the illumination beam 12. The illumination beam 12 thus passes through the beam splitter mirror 14 and strikes a scanning mirror 16. As indicated in FIG. 1 by the double-headed arrow, the scanning mirror 16 can be tilted. The illumination beam 12 is deflected by the tilting movement of the scanning mirror 16 according to the required scanning movement.
After being reflected at the scanning mirror 16, the illumination beam 12 passes through a scanning lens 18 of focal length f3, a tube lens 20 of focal length f2 and an objective 22 of focal length f1. The objective 22 finally focuses the illumination beam 12 onto a sample 24. The focused illumination beam 12 scans the sample 24 point by point by way of the tilting movement of the scanning mirror 16.
A detection beam which is denoted in FIG. 1 by reference numeral 26 and which emanates from a scanned point illuminated by the focused illumination beam 12 arrives back in the objective 22 and passes through the beam path described above in the opposite direction until it strikes the beam splitter mirror 14. Said mirror is configured so that it reflects light of the wavelength of the detection beam 26. The beam splitter mirror 14 thus deflects the detection beam 26 onto a lens 28 which focuses the detection beam 26 onto a confocally arranged pin diaphragm 30. The pin diaphragm 30 filters out of the detection beam 26 all the light originating from regions of the sample 24 which originates outside the light spot generated on the sample 24 by the illumination beam 12. The light which passes through the pin diaphragm 30 finally arrives at an image sensor 32 which can be read out via a controller 34. The light emanating from the sample 24 can thus be detected for each individual scanned point and the detection signal thus generated can be composed into an image.
An essential feature of the confocal microscope 10 according to FIG. 1 in the present context is that the detection beam 26 emanating from the sample 24 is directed back onto the scanning mirror 16, so that the detection beam 26 is influenced by the scanning mirror 16 in the same way as the illumination beam 12. Consequently, the detection beam 26 strikes the image sensor 32 in a stationary manner, while the illumination beam 12 performs a scanning movement on the sample 24 by way of the scanning movement of the scanning mirror 16. The detection beam 26 is held in a quasi stationary manner on the image sensor 32 by way of the return of the detection beam onto the scanning mirror 16. In this context, “stationary” means that although the incidence angle at which the detection beam 26 strikes the image sensor 32 can vary (in the embodiment according to FIG. 1, this incidence angle is stationary), the location of the light incidence cannot.
The principle of holding the detection beam 26 stationary in the sense explained above on the image sensor 32, by way of returning it onto the scanning element 16, is also known in the present technical field as “descanning”. In order to allow “descanning” of this type, in the confocal microscope according to FIG. 1, the scanning mirror 16 is arranged in a plane 36 which is an optically conjugate plane with respect to the object plane denoted in FIG. 1 by reference numeral 38. FIG. 1 also shows an intermediate image plane 40 and 42. The intermediate image plane 40 corresponds optically to plane 36 and is optically conjugate with object plane 38. The intermediate image plane 42 corresponds optically to object plane 38 and is optically conjugate with plane 36.